The goal of this study is to develop the use of cell free extracts from Micrococcus luteus for the introduction of novel amino acids into proteins. This study will optimize the procedures for incorporation of non-natural amino acids with this organism which contains several unassigned codons with the goal of producing a robust methodology and product line to be used by protein chemists to rapidly and easily incorporate probes into proteins under study. The specific aims are to: (l)Evaluate the efficiency of M. luteus unassigned codons for insertion of unnatural amino acids. (2) Test remaining codons which are unused by M. luteus to determine if they are unassigned. (3) Insert two different non-coded amino acids into different locations in dihydrofolate reductase. (4) Test the ability of E. coli tRNAs to complement M. luteus tRNAs in cell free extracts. (5) Synthesize a number of suitably derivatized non-natural amino acids for attachement to the 3'-end of a tRNA and incorporate them into a protein in the M. luteus extract. The phase I results will guide the development of our product line, which will include the extracts, modified amino acids and tRNAs, and reagents in support of this methodology. PROPOSED COMMERCIAL APPLICATIONS: The development of an in vitro translation kit to allow the incorporation of non-natural amino acids into proteins would find enormous utility in the site specific labeling of proteins, or the design of better enzymes through enhancement of catalytic properties or specificity.